PROJECT SUMMARY The nucleotide excision repair (NER) pathway is the main pathway responsible for the removal of bulky DNA lesions such as those formed by UV irradiation. It is highly conserved and its deregulation is linked to human disorders such as xeroderma pigmentosum. In cells, NER does not occur on naked DNA templates, but on a DNA-protein complex called chromatin. The integrity and the structure-dynamics of chromatin are fundamental to the normal function and survival of all eukaryotes. Changes in chromatin architecture directly influence genome accessibility and thus nuclear processes such as DNA damage repair via NER. Yet our knowledge of how NER occurs in its native environment, chromatin is limited. Emerging evidence indicates the important role of two key chromatin regulator in modulating chromatin dynamics to facilitate damage repair in NER. These two regulators are the INO80 ATP- dependent chromatin remodeler and the heterochromatin protein 1. More importantly, the functions of these proteins in NER appear to be at least in part due to their ability to interact with either NER repair factors (e.g. UV-DDB complex) and/or chromatin structural components (e.g. histone variant H2A.Z). To elucidate the molecular mechanism of chromatin structure-dynamics regulation and how it is coupled to DNA damage repair, we will use a combination of single-particle cryo-electron microscopy (EM), traditional biochemistry and biophysical techniques to address the following questions: (1) what is the structural basis of chromatin folding mediated by histone variant H2A.Z? (2) What is the structural mechanism of chromatin compaction by linker histone H1? (3) How is preexisting chromatin environment impact lesion detection in the NER pathway? (4) Do photo lesions on nucleosome interfere with INO80-mediated ATP-dependent chromatin remodeling and what is the mechanism of INO80 recruitment to photo lesions? (5) What is the molecular and structural mechanism of HP1 targeting to photo lesion on chromatin? Overall, this work will greatly improve our understanding of the role chromatin play in genome surveillance system. It will also provide critical insights into how regulation of chromatin structure-dynamics via either histone proteins or chromatin associated factors influence key steps during repair.